Tryptophan dioxygenase and Indoleamine 2,3-dioxygenase: binding and catalysis, a combined simulation and experimental study

LUCIANA CAPECE; ARIEL LEWIS-BALLESTER; SYUN-RU YEH; DARIO A. ESTRIN; MARCELO A. MARTI

Parma

Congreso; XVII Oxygen Binding and Sensing Proteins Meeting; 2012

University of Parma

Tryptophan dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) are the only two heme proteins that catalyze the oxidation reaction of tryptophan (Trp) to N- formylkynurenine. Human IDO (hIDO) has recently been recognized as a potent anticancer drug target, a fact that triggered intense research on the reaction and inhibition mechanisms of hIDO. In this work we have applied a combination of simulation and experimental techniques to study ligand binding, the reaction mechanism, and stereoselectivity of hIDO and xcTDO. Our results revealed that the dioxygenase reaction catalyzed by hIDO and TDO is initiated by addition of the ferric iron-bound superoxide to the C(2)═C(3) bond of Trp to form a ferryl and Trp-epoxide intermediate, via a 2-indolenylperoxo radical transition state. The data demonstrate that the two atoms of dioxygen are inserted into the substrate in a stepwise fashion, challenging the paradigm of heme-based dioxygenase chemistry. Lately, the second ferryl oxygen is inserted into the Trp-epoxide to form the NFK product, involving proton transfer from Trp-NH(3)(+) to the epoxide oxygen, triggering epoxide ring opening and a concerted nucleophilic attack of the ferryl oxygen to the C(2) of Trp that leads to a metastable reaction intermediate. This intermediate subsequently converts to NFK, following C(2)-C(3) bond cleavage and the associated back proton transfer from the oxygen to the amino group of Trp. Regarding stereoselectivity, it is known that while human IDO is able to oxidize both L- and D-Trp, human TDO (hTDO) displays major specificity for L-Trp. By means of molecular dynamics simulation studies combined with muthagenesis experiments, we showed that Thr342 in hTDO is able to regulate the stereoselectivity in TDO. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme but totally abolishes the substrate stereoselectivity.